Tumor Necrosis Factor (TNF) is a cytokine with diverse biological effects which are mediated by binding to specific cell surface receptors. TNF is a potent activator of granulocytes, monocytes, and eosinophils. This agent is also a direct mediator of macrophage cytotoxicity. TNF induces alterations in endothelial cell surface antigen expression, stimulates growth in diploid fibroblasts and promotes the release of a variety of other mediators including GM-CSF, IL-1, and prostaglandins. Moreover, TNF acts as an inducer of monocytic differentiation in HL-60 promyelocytic leukemia cells. TNF is directly cytotoxic for some but not all epithelial tumor cells in vitro. Normal cells and some tumor cells are resistant to TNF despite the presence of TNF receptors. The mechanisms responsible for TNF cytotoxicity and resistance are not known. We have shown that some epithelial tumor cells resistant to TNF induced cytotoxicity produce both TNF transcripts and TNF protein. Furthermore, we have shown that TNF sensitive ZR-75-1 breast carcinoma cells exposed to TNF produce TNF protein. Prolonged exposure to TNF results in stable TNF production and resistance. These studies suggest that TNF may act as an autocrine factor in certain systems. This proposal will focus on the intracellular events which follow TNF/receptor binding. The proposed work will examine three related models: 1) TNF induced HL-60 differentiation; 2) TNF cytotoxicity in epithelial cells; and 3) TNF resistant epithelial cells which produce TNF. In each model, we will examine 1) G protein effects including GTP binding and GTPase activity; 2) inositol formation; 3) Alterations in Ca++ fluxes and cytosolic Ca++ concentrations; 4) Arachidonic acid derived messenger release; and 5) Protein Kinase C activation. These effects will be correlated with TNF induced changes in gene expression, DNA synthesis, phenotype and cytolysis. Together, these studies will increase our understanding of TNF action and the interrelated signal transduction pathways which are involved. The specific aims of this proposal are: 1) To investigate signal transduction during TNF induced monocytic differentiation in HL-60 cells, including G-proteins, phosphatidylinositols, Ca++, arachidonic acid metabolism, protein kinase C activation and gene expression; 2) To examine these same systems in TNF signal transduction in epithelial tumor cells and its relationship to cytotoxicity and TNF resistance; 3) To explore effects of TNF on diploid fibroblasts and its relationship to TNF expression, cell replication and alterations in post receptor binding events.